Overview
Goal is to generate PCR fragments that contain the ends of PAC inserts that can be sequenced. For inverse PCR we cut the PAC once in the vector (near outward primer site) and once at unknown site in insert (and many other places outside these sites, that don't matter). Following ligation to generate small circles, PCR is performed with outward facing primers at the end of the vector that will amplify a product that contains the end of the insert. To increase the odds of getting an amplifiable product, we use two different enzymes (used singly!!) per pac end - NlaIII and RsaI for the SP6 end, and NlaIII and HinPI (or HhaI) for the T7 end.
Details
Details of this protocol, Inverse PCR For PAC-End Sequencing, are located on a web site other than Biocompare Protocols.